Cytochrome c oxidase, the terminal protein of the electron transport chain, is composed in mammals of thirteen subunits, three encoded by mitochondrial DNA and the remainder by nuclear DNA. At least three of these nuclear subunits occur in more than one molecular form, isoforms, the products of separate genes. The proposed experiments will provide insights into the cellular function of these isoforms, which may be related to both ontogenetic development and cellular physiology. In addition, isoform expression has been implicated in human neuromuscular disease. This project will focus on two subunits, COX VIIa and COX VIIc, encoded by three genes. COX7a displays a heart- and muscle-specific isoform gene, COX7a-H, which is already isolated, and an isoform gene expressed in all tissues, COX7a-L. A third gene, COX7c, appears to have a single expressed form in all tissues. The specific aims are to (1) isolate and characterize the COX7a-L and COX7c genes, and compare the regulatory regions to those of COX7a-H; (2) make reporter constructs of each gene to define cis-acting regulatory elements by deletional and mutational approaches; (3) investigate the effect of physiological conditions on regulation of isoform expression; and (4) isolate and study trans-acting regulatory factors. The third aim, studying regulation of isoform expression, will be pursued in two ways: by in situ hybridization of isoform-specific probes to regions of the heart that operate under very different oxidative stresses, and by cotransfection of both isoform genes in distinguishable reporter constructs into cells placed under a variety of physiological conditions. This project fits into the longer term objectives of deducing the role of the nuclear- encoded subunits and the mechanisms used to regulate oxidative function, and applying this knowledge to the study of nuclear genes in mitochondrial disease.